NMR Analysis of Pressure-Induced Structural Changes in Prioteins

1.利用自行研制的高压核磁共振技术，研究了压力对溶液中蛋白质结构的影响。这项技术在现代高场核磁共振波谱仪上对溶液中的蛋白质进行操作，压力在1到2000巴之间，频率为17.6T或750 MHz，持续时间为1H.2。我们将这一技术应用于许多蛋白质，包括15N-统一标记的BPTI(碱性胰蛋白酶抑制物)、细菌视紫红质的螺旋(1-36)、HPR(含组氨酸的磷酸载体蛋白)、Ralgef-RBD(Ral鸟嘌呤核苷酸交换因子-RAS结合域)。在多肽NH的15N/1H二维光谱中揭示了压力诱导的1H和15N化学位移变化，表明折叠蛋白质的整个骨架结构都对压力做出响应，但这种响应在序列上是不均匀的。从压力诱导的化学位移，我们得出结论：(1)氨基NH基团的几乎所有氢键的距离都随着压力而缩短；(2)压力诱导的15N位移报告了多肽主链在压力下的非均匀构象变化。从压力诱导的BPTI的NOE(核Overhauser效应)强度的变化，我们得出结论，压力诱导蛋白质某些区域的选择性压缩，并伴随着一些芳香环的触发器运动减慢。最后，从Ralgef-RBD的信号强度测量中，我们发现压力优先诱导蛋白质在1000bar以上的环区局部展开，然后在2000bar处诱导整个蛋白质的展开。

1. We utilized a novel high pressure NMR technique that was developed in our laboratory to study the effect of pressure on protein structures in solution. The technique works on proteins in solution at any pressure between 1 and 2000 bar on a modern high field NMR spectrometer operating at 17.6 T or 750 MHz for 1H.2. We applied this technique to a number of proteins including 15N -uniformly labeled BPTI (basic pancreatic trypsin inhibitor), helix (1-36) of Bacteriorhodopsin, HPr (histidine-containing phospho-carrier protein), RalGEF-RBD (Ral guanine-nucleotide exchange factor-Ras binding domain). Pressure-induced 1H and 15N chemical shift changes revealed in 15N /1H two-dimensional spectra of peptide NH groups showed that the entire backbone structure of a folded protein responds to pressure, but that the response is non-uniform over the sequence.3. From pressure-induced chemical shifts, we conclude (1) Distances of practically all the hydrogen bonds of amide NH groups, either with carbonyls or solvent water, are shortened by pressure ; (2) Pressure-induced 15N shifts report non-uniform conformational changes in the polypeptide backbone by pressure.4. From pressure-induced changes in NOE (Nuclear Overhauser Effect) intensities for BPTI, we conclude that pressure induces selective compactions of certain regions of the protein, with concomitant slowing down of flip-flop motions of some aromatic rings.5. Finally, from signal intensity measurements in RalGEF-RBD, we found that pressure induces local unfolding of the protein preferentially in loop regions above 1000 bar, followed by unfolding of the entire protein at 2000 bar.